PROJECT SUMMARY Intergenerational obesity and diabetes are at epidemic proportions in the US, with exclusive breastfeeding thought to lessen the risk of obesity and diabetes transmission from mother to child. However, human milk exhibits high individual variability in non-nutritive bioactive compounds and we are now realizing that this variation tracks maternal nutritional status. It is critical to provide a more refined evidence base on the causes and consequences of breast milk variation, especially for infants at high risk of future obesity and diabetes. In our current grant period we are advancing the field of human milk research to show that milk hormone and cytokine concentrations, as well as novel milk metabolomic signatures, significantly vary with maternal body weight and adiposity, and that these milk ?bioactives? predict altered early infant growth. The successful first 5 years of the MILk cohort (Mothers and Infants Linked for Healthy Growth), composed of 360 exclusively breastfeeding, non-diabetic mother-infant dyads, is one of very few studies that are equipped to both comprehensively characterize milk variation in a large sample of deeply phenotyped women, and also to examine prospective associations in their children. The primary objectives of the proposed renewal application are: 1) to expand our understanding of the causes and consequences of breast milk variation for women and children from birth to age 5 years. The Specific Aims are 1) To identify human milk bioactives that differ by maternal pregnancy metabolic and weight status; 2) To identify human milk bioactives that are associated with infant and early childhood growth and metabolic outcomes; and 3) To test changes during lactation in the metabolomic profile of human milk in women with and without GDM. These aims will be achieved by leveraging the resources of the existing MILk Study cohort and milk specimens, and by expanding enrollment to additional women with GDM, with a particular emphasis on enrolling and retaining women of color. The study uses state-of-the-science infant body composition methods, evaluating energy expenditure and fuel oxidation in the offspring while employing comprehensive breast-milk sampling techniques and metabolomic analysis. The proposed research is significant because it tackles an understudied, but potentially important pathway explaining the vicious cycle of maternal-child obesity/diabetes transmission prevalent today. The results of the study will help to design interventions to optimize milk composition in, and provide tailored lactation support for, women with obesity and diabetes. The ultimate goal is to support breastfeeding women and their infants during the first 1000 days of the child?s life.